Project Summary/Abstract The Hedgehog (Hh) cell?cell signaling pathway plays key roles in embryonic development, in maintenance of adult stem cells, and, when improperly regulated, in various birth defects and cancers. In vertebrates, the prototypical activator of the Hh pathway is the secreted protein Sonic hedgehog (SHH), which must travel far from the cells that produce it (?sending cells?) to the cells to which it signals (?receiving cells?). Paradoxically, though, SHH is covalently attached to cholesterol, a modification that tethers it to the plasma membrane of sending cells. Recent studies discovered that the membrane protein Dispatched1 (DISP1) and the SCUBE family of secreted proteins are required for SHH release from sending cells. However, the precise mechanism through which SCUBE2 drives SHH release is unknown. Furthermore, it is unknown whether SCUBE2 actively contributes to SHH delivery to receiving cells, where SHH must bind to its receptor, Patched1 (PTCH1), and at least one of three functionally redundant co-receptors, CDO, BOC, and GAS1. My preliminary data demonstrates that: (1) SCUBE2 can release a heterologous cholesterol-modified protein, but less efficiently than it releases SHH; (2) SCUBE2 directly enhances SHH delivery to receiving cells; (3) SCUBE2 binds to CDO; and (4) different parts of SCUBE2 are involved in SHH release and SHH delivery. These data suggest that (1) SCUBE2 drives SHH release by first binding SHH protein on sending cells and subsequently binding its cholesterol anchor, and (2) SCUBE2 facilitates SHH delivery to receiving cells by directly binding SHH co-receptors. The present proposal aims to: (1) determine how SCUBE2 recognizes the cholesterol anchor of SHH, and to test the function of cholesterol-binding in SHH release from cells, using photocrosslinking with novel photoreactive cholesterol analogs and mass spectrometry; (2) elucidate how SCUBE2 selectively releases SHH from cells, using live-cell fluorescence microscopy and release assays; and (3) elucidate the mechanism and function of interaction between SCUBE2 and SHH co-receptors in SHH delivery, using live-cell fluorescence microscopy and knockout and overexpression approaches in cell-based signaling assays. These experiments will clarify how membrane-anchored SHH is released and delivered to distant cells, providing critical missing information in our understanding of Hh signaling. This information, in turn, will define novel molecular targets, at the level of ligand release and delivery, for blocking aberrant Hh signaling in cancer.